The overall goal of this proposal is to obtain an in depth understanding of the mechanistic links between alterations of the dystrophin glycoprotein complex (DGC) and the impairemnt in the calcium (Ca2+) release mechanisms observed in muscle fibers from various dystrophic animals which serve as models for human muscular dystrophies. Ca2+ release evoked by action potentials (APs) or voltage clamp pulses is significantly impaired in muscle fibers from adult mdx and transgenic sarcospan (SSPN) overexpressing phenotypic (SSPN-Tg) mice. The emerging picture is that the limitations in the Ca2+ release observed in adult dystrophic muscle fibers may result from a suboptimal molecular organization of the underlying molecular machinery supporting the excitation-contraction coupling (ECC) process due to a defective DGC. We hypothesize that the proper organization of the DGC is crucial not only to maintain the sarcolemmal integrity of the muscle fibers during mechanical activity, but also to provide structural and functional support for the transverse tubular system (TTS) and internal membrane compartments which are directly responsible for the ECC process. The specific goals of the application will be to first investigate the molecular mechanisms responsible for the impairment of Ca2+ release in mdx mice (Aim 1), an animal model that shares with Duchenne Muscular Dystrophy (DMD) the absence of dystrophin in the DGC in which most of the advances in muscular dystrophy research have been attained. However, since the pattern of phenotypic alterations in mdx mice results in a relatively benign pathology, possibly due to utrophin substitution in the DGC, experiments will be also carried out in double knockout mdx/utrophin (mdx/utr-/-) mice that display a phenotype comparable to that in DMD patients (Aim 2). We will also use transgenic animal models with other genetic conditions altering the DGC (SSPN-Tg, Utr-TET and others) in order to compare their limitations in the EC coupling with respect to control and mdx mice (aim 2). The last goal of the proposal is to investigate in live muscle fibers whether the association of the different protein components of the DGC is relegated to the sarcolemma or if they interact with the TTS and the sarcoplasmic reticulum (SR), such that alterations in the DGC may ultimately explain the limited functionality of the EC coupling process (Aim 3). The investigations will be carried out using electrophysiological methods, state-of-the-art high-resolution optical methods (FRET and TIRFM) that permit the assessment of the localized expression in live cells of protein components of the DGC and other key muscle proteins involved in EC coupling, and their role in muscle function at the cellular, single sarcomere, and molecular level.